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Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs, with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to 18.98 μg/mg and 9.15 μg/mg (dry weight), respectively. Importantly, eicosapentaenoic acid (EPA) was accumulated up to a highest record of 85.35 μg/mg by metabolic engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas DHA was found exclusively in phospholipids. Combinatorial expression of these critical enzymes led to the optimal increment of LC-PUFAs without unbalanced metabolic flux and demonstrated the practical feasibility of generating sustainable LC-PUFA production.

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.

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